No description available.

To study adaptation of pancreatic amylase to diet, an affinity adsorbent, alpha-GHI-AH-Sepharose 4B, was used to determine amylase synthesis in cultured pancreatic acinar cells. This adsorbent exhibited a consistent binding capacity and was specific for amylase. Acinar cells from rats fed high fat (HF) or carbohydrate (HC) diets for 7 d were cultured 1-48 h in serum-free medium. Amylase activity remained significantly higher in HC cells than in HF cells through 24 h in culture, despite its decrease with time in culture. The relative synthesis of amylase (3H-phe amylase/3H-phe total protein x 100) was significantly higher in HC than in HF cells at isolation and remained higher during culture. These results demonstrate that this affinity adsorbent can be used to determine amylase synthesis and suggest that the effect of diet on amylase activity and relative synthesis persists in cultured pancreatic acinar cells.

Type 1 diabetes (T1D) is a chronic disease, characterised by the destruction of pancreatic beta cells, which results in lack of insulin expression. Most current therapies rely on the exogenous administration of recombinant insulin. Islet transplantation has been shown to be a more effective alternative treatment, but it is also limited by the lack of available islets for transplantation. The recently awarded work of Yamanaka and colleagues has shown that fully differentiated cells can be reprogrammed towards their pluripotent, undifferentiated state, through overexpression of a simple combination of four key transcritption factors (TFs). The studies presented in this thesis sought to investigate whether a combination of a small group of key pancreatic TFs would be able to drive both mouse embryonic stem cells (mES) and fully differentiated rat and human acinar cells of the pancreas towards insulin-producing cells. When administered in a timely manner to mES cells, the pancreatic TFs Pdx1 and MafA were able to induce the formation of cells that synthesised insulin de novo. Further studies aimed at investigating if a small number of TFs would be able to reprogramme the exocrine pancreatic cells towards insulin expressing cells, since, unlike endocrine cells, exocrine cells are highly abundant in the pancreas. Studies performed in both a rat exocrine cell line (AR42J-B13) and in human exocrine cells demonstrated that the combination of the TFs Pdx1, MafA, Ngn3 and Pax4 was able to generate glucose responsive β-like cells in both models. In addition, Pax4 was found to be determinant for the functionality of the generated β-cells. The functionality of these cells was further demonstrated by their ability to prevent the onset of hyperglycemia upon transplantation into a diabetic mouse model. The work presented in this thesis has shown that cultured exocrine cells may be a promising alternative for generating a replenishable supply of β-cells for transplantation.

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd deadliest cancer in the United States with a projected 12% 5-year survival rate. Acinar cells have been proposed as a potential cell-of-origin for PDAC after undergoing acinar to ductal metaplasia (ADM). In the absence of oncogenic mutations (e.g. Kras), ADM lesions form as an adaptive response and eventually resolve to regenerate the acinar compartment, which we term as adaptive ADM. However, in the presence of oncogenic Kras mutations, the ADM lesions can transform to a pre-invasive state called pancreatic intraepithelial neoplasia (PanIN). Thus, a normally adaptive metaplastic response becomes maladaptive, which we term as oncogenic ADM. The mechanisms that drive PanIN formation in the context of injury and oncogenic mutations are poorly understood, resulting in an absence of targets to combat persistent ADM. This thesis investigates the role of FRA1 (gene name Fosl1) in acinar cell de-differentiation, PanIN transformation, and eventual PDAC tumorigenesis. Through CUT&RUN sequencing of mice undergoing recovery from caerulein-induced acute pancreatitis, we identify FRA1 as the most active transcription factor during KrasG12D mediated acute pancreatitis- mediated injury. We have elucidated a functional role of FRA1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D. Using a gene regulatory network and pseudotime trajectory inferred from single nuclei ATAC-seq and bulk-RNA seq, we hypothesize a regulatory model of the acinar-ADM-PanIN continuum and experimentally validate that Fosl1 knockout mice are delayed in the onset of ADM and PanIN. Furthermore, deletion of Fosl1 in an autochthonous PDAC mouse model revealed that this ADM-initiated delay eventually culminates in a significant survival advantage and a less aggressive tumor phenotype. Through investigation of upstream regulators of FRA1, we identified G-CSF as an ADM-promoting cytokine. Fosl1 depletion prevented the pro-inflammatory effects of G-CSF, indicating that the G-CSF/FRA1 signaling axis can modulate ADM. Using ex vivo acinar cultures, we also showed that G-CSF can induce FRA1 through MEK/ERK signaling. Our findings reveal that FRA1 is a mediator of acinar cell plasticity and contributes to acinar cell de-differentiation and malignant transformation. Although the majority of this thesis focuses on oncogenic ADM, we also include a chapter on the role of Prrx1 in adaptive ADM. Our comprehensive and unbiased approach identified previously the Paired-Related homebox1 (Prrx1) as the most upregulated transcription factor in the intersection of pancreatic ductal development, regeneration, and evolution of PanIN. We have demonstrated previously that Prrx1 can promote a ductal phenotype by binding the Sox9 promotor and inducing its expression during pancreatitis. In this body of work, we present a novel mechanism by which Prrx1 regulates maintenance of adaptive ADM. Using novel mouse models and ex vivo acinar culture systems, we demonstrate that Prrx1 can induce TGFβ signaling and reduce E-Cadherin expression to promote ADM. We do not know if there is any potential epistatic interaction between FRA1 and PRXX1. Overall, we reveal the rippling effects of FRA1 can have during the early stages of pre-neoplasia, and we unveil an alternative function of PRRX1 for stimulating an adaptive response to stress. This thesis presents a new understanding of how acinar cell de-differentiation occurs in the pancreas by revealing novel roles of two transcription factors, FRA1 and PRRX1, and furthers our understanding of tissue regeneration in an injured pancreas.

Receptor-activated phosphatidylinositol (PtdIns) hydrolysis was examined in AR42J rat pancreatic acini. Cholecystokinin-octapeptide (CCK₈) and bombesin induced a dose-dependent accumulation of [³H] inositol monophosphate ([³H]InsP₁). Manganese (Mn²⁺), a known calcium channel blocker, did not alter basal PtdIns hydrolysis. In contrast, when added 5 minutes prior to the addition of agonists for 60 minutes, Mn²⁺ markedly inhibited secretagogue-mediated [³H]InsP1 formation. Mn²⁺ also attenuated the CCK₈-mediated increase in biologically active inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. These inhibitory effects of Mn²⁺ were mimicked by lanthanum and by EGTA. Addition of calcium to EGTA-treated cells abolished the inhibitory effects of extracellular calcium depletion. Mn²⁺, La³⁺ and EGTA exerted similar inhibitory effects on PtdIns hydrolysis in pancreatic acini. These findings suggest that receptor-activated calcium influx is required for full activation of the CCK₈-mediated signal transduction pathway that is coupled to PtdIns hydrolysis.

Pancreatic acinar cells display plasticity to acquire distinct differentiation programs, being involved in diseases as chronic pancreatitis and pancreatic ductal adenocarcinoma. This work shows that acinar cells cultured in suspension undergo dedifferentiation, acquiring a pancreatic embryonic progenitor phenotype. Dedifferentiated cells turn on a senescent program, associated with activation of p53 and Ras pathways. A similar progenitor‐like phenotype with activation of senescence is present in experimental chronic pancreatitis. Acinar cultures lacking p53 overcome growth arrest and lose the pancreatic phenotype, undergoing an epithelial‐mesenchymal transition, while maintaining the expression of pre‐pancreatic endoderm and stem cell markers. In experimental acute pancreatitis, absence of p53 results in increased acinar cell proliferation and delayed regeneration. Our findings support a role for acinar cell dedifferentiation in the initiation of pancreatic diseases. A p53‐ dependent control of cell growth and epithelial differentiation constitutes a tumor suppressive mechanism that may limit PDAC development. / Las células pancreáticas acinares poseen plasticidad que les permite adquirir distintos programas de diferenciación, estando implicadas en enfermedades como la pancreatitis crónica y el adenocarcinoma ductal pancreático. En este trabajo hemos demostrado que las células acinares cultivadas en suspensión se desdiferencian, adquiriendo un fenotipo de progenitores pancreáticos embrionarios. En estas células se induce un programa de senescencia asociado con la activación de las vías de p53 y Ras. Un fenotipo similar se evidencia en modelos de pancreatitis crónica experimental. Cultivos acinares en los que se ha inactivado p53 sobrepasan el bloqueo de crecimiento y pierden el fenotipo pancreático, presentando una transición epitelio‐mesenquimal y manteniendo la expresión de marcadores de endodermo pre‐pancreático y de células madre. Durante la inducción de una pancreatitis aguda experimental, la ausencia de p53 resulta en un incremento de la proliferación acinar y en un retraso en la regeneración. Nuestros resultados demuestran que la desdiferenciación de las células acinares participa en el desarrollo de enfermedades pancreáticas. El control del crecimiento celular y de la diferenciación pancreática epitelial dependiente de p53 constituye un mecanismo de supresión tumoral que puede limitar el desarrollo del PDAC.

A pesar de la abundancia de estudios realizados sobre el papel de las células acinares en las patologías exocrinas del páncreas (i.e. pancreatitis y cáncer), el estudio de las modificaciones producidas durante la diferenciación acinar en dichas patologías, se ha visto limitado por la escasez de modelos celulares no tumorales. Resultados previos de nuestro laboratorio, muestran que las células mES (células madre embrionarias de ratón )- pluripotentes y con la capacidad para generar tipos celulares especializados- pueden desarrollar un fenotipo acinar in vitro. Los objetivos de esta tesis han sido aumentar el contenido de enzimas digestivos así como las propiedades funcionales de las células generadas. Para ello se sobreexpresaron de forma estable p48, RBPJL y Mist1en células madre por transducción lentiviral de estos genes. Obtuvimos, gracias a una estrategia de infección en múltiples etapas, líneas celulares transgénicas mES que expresaban de forma constitutiva RBPJL y/o Mist1. La superimposición de la expresión de p48 por infección lentiviral en células en proceso de diferenciación dio lugar a una fuerte expresión de enzimas digestivos, con un patrón de regulación similar al que acontece in vivo durante el desarrollo pancreático. En esta inducción, tanto p48 como RPBJL son indispensables. Por otro lado, hemos mostrado un aumento elevado en la producción de varios componentes de la maquinaria secretota dependiente de Mist1. Además, hay que hacer notar ,que las células p48/RBPJL/Mist1 exhiben una regulada-secreción en respuesta a los secretagogos acinares y una mejor actividad de que la línea celular acinar 266-6. La expresión combinada de genes clave implicados en el desarrollo pancreático en células ES es un prometedor abordaje que nos llevará a una comprensión de los sutiles procesos del desarrollo exocrino pancreático. / Despite known involvement of acinar cells in pancreatic exocrine pathologies (i.e pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. Our previous data showed that mES (murine embryonic stem) cells, which are pluripotent and have the ability to generate specialized cell types, can acquire an acinar phenotype in vitro. The aim of this work was to increase the digestive enzyme content of the generated cells as well as their functional properties based on stable overexpression of p48, RBPJL and Mist1 by lentiviral gene transduction. Thus, we engineered transgenic mES cell lines constitutively expressing RBPJL and/or Mist1 using a multi-step infection strategy. The superimposition of p48 expression by lentiviral infection of differentiating cells resulted in a strong expression of digestive enzymes, with a pattern of regulation similar to what occurs in vivo during pancreatic development. In this induction, both p48 and RPBJL are indispensable. On the other hand, we showed a high increase in the production of several components of the secretory machinery which was dependent of Mist1. Importantly, p48/RBPJL/Mist1 cells exhibited a regulated-secretory in response to acinar secretagogues and a better secretion activity than the 266-6 acinar cell line. Combined expression of key genes involved in pancreatic development in ES cells may be a promising approach to better understand subtle steps of pancreatic exocrine development.

Tot i que es coneix l’involucrament de les cèl·lules pancreàtiques acinars en patologies exocrines (pancreatitis i càncer de pàncrees), la manca de models normals basats en cèl·lules ha limitat l’estudi de les alteracions que succeeixen en el programa de diferenciació pancreàtica. Hem demostrat prèviament que les cèl·lules mare embrionàries murines, que són pluripotents, poden adquirir un fenotip acinar in vitro. Això es va aconseguir, en part, amb una combinació de senyals que provenien del cultiu de pàncrees fetals que no era, però, específic del llinatge pancreàtic. L’objectiu d’aquest treball ha estat el de desenvolupar un protocol selectiu pel llinatge acinar basat en l’activació seqüencial de vies de senyalització que recapitulin el desenvolupament pancreàtic in vivo, a través de la formació definitiva de l’endoderm, l’especificació pancreàtica i acinar i l’expansió/diferenciació de progenitors acinars. El tractament de cossos embrionaris amb Activina A va promoure l’expressió de gens d’endoderm com està prèviament descrit. El tractament subsegüent amb àcid Retinoic, FGF10 i Ciclopamina, un inhibidor de la via de Hedgehog, va resutar en la inducció dels marcadors de progenitors pancreàtics Pdx1, Ptf1a i Cpa1 però també d’aquells expressats en el llinatge pancreàtic, que van ser reduïts amb la inhibició de BMPs. Les cèl·lules van ser a continuació cultivades en Matrigel utilitzant un sistema de cultiu en 3D en presència de fol·listatina, dexametasona i KGF comportant una inducció significativa dels nivells de mRNA i proteïna de marcadors acinars i una disminució de l’expressió dels de marcadors acinars. A més, es va veure que Amyl es secretava en el medi. Aquestes dades indiquen que l’activació selectiva del programa de diferenciació acinar en cèl·lules mare embrionàries es pot dur a terme mitjançant una inducció esgraonada de vies de senyalització involucrades en el desenvolupament pancreàtic exocrí proporcionant una eina potencial per estudiar la diferenciació pancreàtica i malalties relacionades amb el pàncrees. / Despite known involvement of pancreatic acinar cells in exocrine pathologies (pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. We have previously shown that mESC (murine embryonic stem cells), which are pluripotent, can acquire an acinar phenotype in vitro. This was achieved, in part, by a combination of signals provided by the culture of foetal pancreases which was, however, no specific for the acinar lineage. The aim of this work was to develop a protocol selective for the acinar lineage based on the sequential activation of signaling pathways that recapitulate pancreatic development in vivo, through the definitive endoderm formation, the pancreatic and acinar specification and the expansion/differentiation of acinar progenitors. Treatment of embryoid bodies with Activin A enhanced the expression of endodermal genes as previously described. Subsequent treatment with Retinoic acid, FGF10 and Cyclopamine, an inhibitor of the Hedgehog pathway, resulted in the enhancement of pancreatic progenitor markers Pdx1, Ptf1a and Cpa1 but also of those expressed in the hepatic lineage, which were reduced by BMPs inhibition. Cells were further cultured in Matrigel using a 3D culture system in the presence of follistatin, dexamethasone, and KGF leading to a significant enhancement of the mRNA and protein levels of acinar markers while decreasing the expression of endocrine ones. Moreover, active Amyl was released into the medium. These data indicate that the selective activation of the acinar differentiation program in ES cells can be achieved by stepwise induction of signaling pathways involved in pancreatic exocrine development providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.